一种快速检测乙型脑炎病毒滴度的荧光灶试验方法的建立、验证及应用  

Establishment,validation and application of fluorescence focus assay for rapid titration of Japanese encephalitis virus

作　　者：罗娜 乔建 姜志军 杨禅堂 张仲锴 吴杰 徐葛林 Luo Na;Qiao Jian;Jiang Zhijun;Yang Shantang;Zhang Zhongkai;Wu Jie;Xu Gelin(Department of Rotavirus Vaccine,Wuhan Institute of Biological Products Co.Ltd,Wuhan 430207,China)

机构地区：[1]武汉生物制品研究所有限责任公司轮状病毒疫苗室,武汉430207

出　　处：《中华微生物学和免疫学杂志》2022年第3期224-233,共10页Chinese Journal of Microbiology and Immunology

摘　　要：目的:建立快速荧光灶试验(fluorescence focus assay,FFA)滴定乙型脑炎病毒(Japanese encephalitis virus, JEV)的方法,验证该法替代病毒蚀斑法测定乙型脑炎减毒活疫苗(简称乙脑减毒活疫苗)滴度的可行性并将该法作初步应用。方法:利用原核表达方式构建JEV非结构蛋白1(non-structural protein 1,NS1)重组体系,以纯化所得的JEV-NS1蛋白免疫家兔制备抗NS1蛋白多克隆抗体,建立快速FFA测定JEV滴度的方法。通过与病毒蚀斑法作比对分析,对其专属性、准确度、精密度、线性、范围和耐用性进行验证。用经验证后的FFA检测28批乙脑减毒活疫苗,分析其应用于生产的可行性。结果:所制备的多克隆抗体与JEV可产生特异性荧光灶反应,与其他病毒(森林脑炎病毒、黄热病毒、人柯萨奇病毒A2型和A4型)无交叉反应;FFA方法学验证结果均符合乙脑减毒活疫苗的质控要求;与蚀斑法比较,FFA检测结果更趋一致。结论:本研究建立的FFA通过方法学验证,可应用于乙脑减毒活疫苗成品的滴度测定。Objective To establish and validate a fluorescence focus assay(FFA)for rapid titration of Japanese encephalitis virus(JEV)and to evaluate its application in the production of Japanese encephalitis vaccine.Methods Recombinant JEV non-structural protein 1(NS1)was expressed in a prokaryotic expression system.After purification,JEV-NS1 was used to immunize rabbits to induce polyclonal antibody.FFA was established with the polyclonal antibody to titer JEV.The accuracy of FFA was validated by comparing with plaque assay,and the specificity,precision,linearity,range and robustness of FFA were also validated.Twenty-eight batches of live-attenuated Japanese encephalitis vaccine were titrated with FFA and plaque assay to analyze the relationship between the two assays.Results FFA established with polyclonal antibody against JEV-NS1 could be used to titrate JEV,and there was no cross reaction with other viruses(tick-borne encephalitis virus,yellow fever virus,coxsackievirus A2,coxsackievirus A4).Results of the validation tests showed that FFA met the requirement of quality control for live-attenuated Japanese encephalitis vaccine.FFA was more consistency than plaque assay.Conclusions The established FFA could be used for virus titration in the production of live-attenuated Japanese encephalitis vaccine.

关 键 词：乙型脑炎减毒活疫苗 多克隆抗体 荧光抗体技术 病毒蚀斑法 验证 

分 类 号：R392-33[医药卫生—免疫学]